Abstract
Background: Cryopreservation is a routinely used methodology for prolonged storage of viable cells. The use of cryo-protective agents (CPAs) such as dimethylsulfoxide (DMSO), glycerol, or trehalose is paramount to reducing cellular cryo-injury, but their effectiveness is still limited. The current study focuses on establishing and modulating the proteomic and the corresponding biological profiles associated with the cryo-injury of human leukemia (HL-60) cells cryopreserved in DMSO alone or DMSO +/-novel CPAs (e. g., nigerose [Nig] or salidroside [Sal]). Findings: To reduce cryo-damage, HL-60 cells were cultured prior and post cryopreservation in malondialdehyde Roswell Park Memorial Institute medium-1640 media +/- Nig or Sal. Shotgun proteomic analysis showed significant alterations in the levels of proteins in cells cryopreserved in Nig or Sal compared to DMSO. Nig mostly affected cellular metabolism and energy pathways, whereas Sal increased the levels of proteins associated with DNA repair/duplication, RNA transcription, and cell proliferation. Validation testing showed that the proteome profile associated with Sal was correlated with a 2.8-fold increase in cell proliferative rate. At the functional level, both Nig and Sal increased glutathione reductase (0.0012 +/- 6.19E-05 and 0.0016 +/- 3.04E-05 mU/mL, respectively) compared to DMSO controls (0.0003 +/- 3.7E-05 mU/mL) and reduced cytotoxicity by decreasing lactate dehydrogenase activities (from -2.5 to -4.75 fold) and lipid oxidation (-1.6 fold). In contrast, only Nig attenuated protein carbonylation or oxidation. Conclusions: We have identified key molecules and corresponding functional pathways underpinning the effect of cryopreservation (+/- CPAs) of HL-60 cells. We also validated the proteomic findings by identifying the corresponding biological profiles associated with promoting an anti-oxidative environment post cryopreservation. Nig or Sal in comparison to DMSO showed differential or additive effects in regard to reducing cryo-injury and enhancing cell survival/proliferation post thaw. These results can provide useful insight to cryo-damage and the design of enhanced cryomedia formulation.